Microbiome variation at the clam-sediment interface may explain changes in local productivity of Chamelea gallina in the North Adriatic sea.

BACKGROUND: The clam Chamelea gallina is an ecologically and economically important marine species in the Northwestern Adriatic Sea, which currently suffers from occasional, and still unexplained, widespread mortality events. In order to provide some glimpses in this direction, this study explores the connections between microbiome variations at the clam-sediment interface and the nutritional status of clams collected at four Italian production sites along the Emilia Romagna coast, with different mortality incidence, higher in the Northern sites and lower in the Southern sites. RESULTS: According to our findings, each production site showed a peculiar microbiome arrangement at the clam-sediment interface, with features that clearly differentiate the Northern and Southern sites, with the latter also being associated with a better nutritional status of the animal. Interestingly, the C. gallina digestive gland microbiome from the Southern sites was enriched in some health-promoting microbiome components, capable of supplying the host with essential nutrients and defensive molecules. Furthermore, in experiments conducted under controlled conditions in aquaria, we provided preliminary evidence of the prebiotic action of sediments from the Southern sites, allowing to boost the acquisition of previously identified health-promoting components of the digestive gland microbiome by clams from the Northern sites. CONCLUSIONS: Taken together, our findings may help define innovative microbiome-based management strategies for the preservation of the productivity of C. gallina clams in the Adriatic Sea, through the identification and maintenance of a probiotic niche at the animal-sediment interface.

Extraction method for the multiresidue analysis of legacy and emerging pollutants in marine mussels from the Adriatic Sea.

The release of hazardous chemicals into aquatic environments has long been a known problem, but its full impact has only recently been realized. This study presents a validated liquid chromatography-mass spectrometry (HPLC-MS/MS) method for detecting pharmaceutical and pesticide residues in mussels (Mytilus galloprovincialis). An innovative MS-compatible extraction method was developed and validated, demonstrating successful recovery rates for analytes at three different concentration levels (25-95%). The method detected the target analytes at ng/g concentrations with high accuracy (-7% to 11%) and low relative standard deviation (<10%) for both intra-day and inter-day analyses. After validation, the method was applied to mussel samples collected from a commercial farm near Senigallia, Adriatic Sea, detecting different contaminants in the range of 2-40 ng/g (dry weight). The study provides a valuable tool for investigating the potential threats posed by diverse contaminant classes with high annual tonnage, including analytes with known persistence and/or illegal status.

Seasonal dynamics of the microbiome-host response to pharmaceuticals and pesticides in Mytilus galloprovincialis farmed in the Northwestern Adriatic Sea.

Marine mussels, especially Mytilus galloprovincialis, are well-established sentinel species, being naturally resistant to the exposure to multiple xenobiotics of natural and anthropogenic origin. Even if the response to multiple xenobiotic exposure is well known at the host level, the role of the mussel-associated microbiome in the animal response to environmental pollution is poorly explored, despite its potential in xenobiotic detoxification and its important role in host development, protection, and adaptation. Here, we characterized the microbiome-host integrative response of M. galloprovincialis in a real-world setting, involving exposure to a complex pattern of emerging pollutants, as occurs in the Northwestern Adriatic Sea. A total of 387 mussel individuals from 3 commercial farms, spanning about 200 km along the Northwestern Adriatic coast, and in 3 different seasons, were collected. Multiresidue analysis (for quantitative xenobiotic determination), transcriptomics (for host physiological response), and metagenomics (for host-associated microbial taxonomical and functional features) analyses were performed on the digestive glands. According to our findings, M. galloprovincialis responds to the presence of the complex pattern of multiple emerging pollutants - including the antibiotics sulfamethoxazole, erythromycin, and tetracycline, the herbicides atrazine and metolachlor, and the insecticide N,N-diethyl-m-toluamide - integrating host defense mechanisms, e.g., through upregulation of transcripts involved in animal metabolic activity, and microbiome-mediated detoxification functions, including microbial functionalities involved in multidrug or tetracycline resistance. Overall, our data highlight the importance of the mussel-associated microbiome as a strategic player for the orchestration of resistance to the multixenobiotic exposure at the holobiont level, providing strategic functionalities for the detoxification of multiple xenobiotic substances, as occurring in real world exposure settings. Complementing the host with microbiome-dependent xenobiotic degradative and resistance genes, the M. galloprovincialis digestive gland associated microbiome can have an important role in the detoxification of emerging pollutants in a context of high anthropogenic pressure, supporting the relevance of mussel systems as potential animal-based bioremediation tool.

Acclimatization of a coral-dinoflagellate mutualism at a CO2 vent.

Ocean acidification caused by shifts in ocean carbonate chemistry resulting from increased atmospheric CO2 concentrations is threatening many calcifying organisms, including corals. Here we assessed autotrophy vs heterotrophy shifts in the Mediterranean zooxanthellate scleractinian coral Balanophyllia europaea acclimatized to low pH/high pCO2 conditions at a CO2 vent off Panarea Island (Italy). Dinoflagellate endosymbiont densities were higher at lowest pH Sites where changes in the distribution of distinct haplotypes of a host-specific symbiont species, Philozoon balanophyllum, were observed. An increase in symbiont C/N ratios was observed at low pH, likely as a result of increased C fixation by higher symbiont cell densities. δ13C values of the symbionts and host tissue reached similar values at the lowest pH Site, suggesting an increased influence of autotrophy with increasing acidification. Host tissue δ15N values of 0‰ strongly suggest that diazotroph N2 fixation is occurring within the coral tissue/mucus at the low pH Sites, likely explaining the decrease in host tissue C/N ratios with acidification. Overall, our findings show an acclimatization of this coral-dinoflagellate mutualism through trophic adjustment and symbiont haplotype differences with increasing acidification, highlighting that some corals are capable of acclimatizing to ocean acidification predicted under end-of-century scenarios.

Glyphosate and its breakdown product AMPA elicit cytoprotective responses in haemocytes of the Mediterranean mussel (Mytilus galloprovincialis).

This study investigates the effects of glyphosate (GLY) and its metabolite AMPA on cytoprotective and detoxification mechanisms in haemocytes of Mytilus galloprovincialis. Cells were treated in vitro with 0.1 and 1.0 µg/L GLY, 0.1 µg/L, 0.1 and 1.0 µg/L AMPA, or two mixtures GLY+AMPA (0.1 µg/L GLY + 0.1 µg/L AMPA, 1.0 µg/L GLY + 1.0 µg/L AMPA). GLY and AMPA increased MXR efflux activity and modulated expression of the ABCB transcript encoding a MXR related ABC transporter P-glycoprotein. The mixtures GLY+AMPA reduced efflux activity with ABCB down-regulation (at 1 µg/L GLY/AMPA). Modulation of lysosomal and immune related transcripts generally agree with known effects of the chemicals on these physiological functions. Given their cumulative action as chemosensitizers of the MXR system, and their interactive effects on haemocyte parameters, glyphosate and AMPA at environmental concentrations should be addressed as a concern factor for the biological vulnerability of marine habitats.

Assessment of bioaccumulation of glyphosate and aminomethylphosphonic acid in marine mussels using capillary electrophoresis with light-emitting diode-induced fluorescence detection.

Glyphosate or N-(phosphonomethyl)glycine, widely used as herbicide in agriculture to control weeds and to facilitate harvesting, has been included in Group 2A pollutants (probably carcinogenic to humans) by the International Agency for Research on Cancer (IARC). In intensive agricultural areas, runoff and soil leaching are likely to drive glyphosate to surface waters, where the compound is often detected together with its main microbial metabolite, aminomethylphosphonic acid (AMPA). In the present study a method based on capillary electrophoresis coupled with light-emitting diode-induced fluorescence detection has been developed and validated for the determination of the two compounds in whole soft mass of marine mussels (Mytilus galloprovincialis). The method is based on the acidic hydrolysis of lyophilized tissue using 6 M HCl (oven at 110 °C for 22 h) to release the target analytes; their subsequent derivatization using 4-fluoro-7-nitro-2,1,3-benzoxadiazole, was found to be suitable for the sensitive fluorescence detection. To achieve optimum separation of the analytes from the matrix and degradation reagent interferences, the background electrolyte constituted by borate buffer (pH 9.2, 30 mM) was supplemented with 10 mM heptakis(2,6-di-O-methyl)-ß-cyclodextrin. The method was validated for linearity, precision, accuracy, robustness and sensitivity showing LOQ of 0.2 and 1.0 µg/g in fresh tissues, for AMPA and glyphosate, respectively; the recovery values ranged within 88.5 - 94.6% for glyphosate and 70.4 - 76.6% for AMPA. Experimental samples of Mediterranean mussels M. galloprovincialis treated with 100 µg/L or 500 µg/L of both glyphosate and AMPA, showed a dose dependent bioaccumulation of the compounds reaching maximum level of 77.0 µg/g and 11.3 µg/g of AMPA and glyphosate, respectively. The study demonstrates for the first time M. galloprovincialis as potential sentinel organisms for the environmental occurrence of these small amphoteric pollutants.

Metagenomic shifts in mucus, tissue and skeleton of the coral Balanophyllia europaea living along a natural CO2 gradient.

Using the Mediterranean coral Balanophyllia europaea naturally growing along a pH gradient close to Panarea island (Italy) as a model, we explored the role of host-associated microbiomes in coral acclimatization to ocean acidification (OA). Coral samples were collected at three sites along the gradient, mimicking seawater conditions projected for 2100 under different IPCC (The Intergovernmental Panel on Climate Change) scenarios, and mucus, soft tissue and skeleton associated microbiomes were characterized by shotgun metagenomics. According to our findings, OA induced functional changes in the microbiomes genetic potential that could mitigate the sub-optimal environmental conditions at three levels: i. selection of bacteria genetically equipped with functions related to stress resistance; ii. shifts in microbial carbohydrate metabolism from energy production to maintenance of cell membranes and walls integrity; iii. gain of functions able to respond to variations in nitrogen needs at the holobiont level, such as genes devoted to organic nitrogen mobilization. We hence provided hypotheses about the functional role of the coral associated microbiome in favoring host acclimatation to OA, remarking on the importance of considering the crosstalk among all the components of the holobiont to unveil how and to what extent corals will maintain their functionality under forthcoming ocean conditions.

Coral micro- and macro-morphological skeletal properties in response to life-long acclimatization at CO2 vents in Papua New Guinea.

This study investigates the effects of long-term exposure to OA on skeletal parameters of four tropical zooxanthellate corals naturally living at CO2 seeps and adjacent control sites from two locations (Dobu and Upa Upasina) in the Papua New Guinea underwater volcanic vent system. The seeps are characterized by seawater pH values ranging from 8.0 to about 7.7. The skeletal porosity of Galaxea fascicularis, Acropora millepora, massive Porites, and Pocillopora damicornis was higher (up to ~ 40%, depending on the species) at the seep sites compared to the control sites. Pocillopora damicornis also showed a decrease of micro-density (up to ~ 7%). Thus, further investigations conducted on this species showed an increase of the volume fraction of the larger pores (up to ~ 7%), a decrease of the intraskeletal organic matrix content (up to ~ 15%), and an increase of the intraskeletal water content (up to ~ 59%) at the seep sites. The organic matrix related strain and crystallite size did not vary between seep and control sites. This multi-species study showed a common phenotypic response among different zooxanthellate corals subjected to the same environmental pressures, leading to the development of a more porous skeletal phenotype under OA.

Variability of metabolic, protective, antioxidant, and lysosomal gene transcriptional profiles and microbiota composition of Mytilus galloprovincialis farmed in the North Adriatic Sea (Italy).

This study evaluates the transcriptional profiles of genes related to physiological responses in digestive glands (DG) of Mytilus galloprovincialis under the influence of seasonal changes of environmental variables, gender bias, and gonadal development. Composition of the DG microbiome was also explored. Mussels were collected across 7 months encompassing 3 seasons from a farm in the Northwestern Adriatic Sea. All gene products showed complex transcriptional patterns across seasons. Salinity, surface oxygen and transparency significantly correlate with transcriptional profiles of males, whereas in females temperature and gonadal maturation mostly explained the observed transcriptional changes. Seasonal variations and gender-specific differences were observed in DG microbiome composition, with variations resembling metabolic accommodations likely facing season progression and reproductive cycle. Results provide baseline information to improve actual monitoring strategies of mussel farming conditions and forecast potential detrimental impacts of climatological/environmental changes in the study area.

Accumulation of PAHs in the tissues and algal symbionts of a common Mediterranean coral: Skeletal storage relates to population age structure.

Polycyclic aromatic hydrocarbons (PAHs) are widespread and harmful environmental pollutants that threaten marine ecosystems. Assessing their level and source is crucial to estimate the potential risks for marine organisms, as PAHs represent an additional threat to organism resilience under ongoing climatic change. Here we applied the QuEChERS extraction method to quantify four PAHs (i.e. acenaphthene, fluorene, fluoranthene, and pyrene) in three biological compartments (i.e. skeleton, tissue, and zooxanthellae symbiotic algae) of adult and old specimens of a scleractinian coral species (Balanophyllia europaea) that is widespread throughout the Mediterranean Sea. A higher concentration of all four investigated PAHs was observed in the zooxanthellae, followed by the coral tissue, with lowest concentration in the skeleton, consistently with previous studies on tropical species. In all the three biological compartments, the concentration of low molecular weight PAHs was higher with respect to high-molecular weight PAHs, in agreement with their bioaccumulation capabilities. PAH concentration was unrelated to skeletal age. Observed PAHs were of petrogenic origin, reflecting the pollution sources of the sampling area. By coupling PAH data with population age structure data measured in the field, the amount of PAHs stored in the long term (i.e. up to 20 years) in coral skeletons was quantified and resulted in 53.6 ng m-2 of acenaphthene, 69.4 ng m-2 of fluorene, 2.7 ng m-2 of fluoranthene, and 11.7 ng m-2 of pyrene. This estimate provides the basis for further assessments of long-term sequestration of PAHs from the marine environment in the whole Mediterranean, given the widespread distribution of the investigated coral species.

Evaluating bivalve cytoprotective responses and their regulatory pathways in a climate change scenario.

Temperature is a relevant abiotic factor affecting physiological performance and distribution of marine animals in natural environments. The changes in global seawater temperatures make it necessary to understand how molecular mechanisms operate under the cumulative effects of global climate change and chemical pollution to promote/hamper environmental acclimatization. Marine mussels are excellent model organisms to infer the impacts of those anthropogenic threats on coastal ecosystems. In this study, Mediterranean mussels (Mytilus galloprovincialis) were exposed to different concentrations of the metal copper (Cu as CuCl2: 2.5, 5, 10, 20, 40 µg/L) or the antibiotic oxytetracycline (OTC: 0.1, 1, 10, 100, 1000 µg/L) at increasing seawater temperatures (16 °C, 20 °C, 24 °C). Transcriptional modulation of a 70-kDa heat shock protein (HSP70) and of the ABC transporter P-glycoprotein (P-gp, encoded by the ABCB gene) was assessed along with the cAMP/PKA signaling pathway regulating both gene expressions. At the physiological temperature of mussels (16 °C), Cu and OTC induced bimodal changes of cAMP levels and PKA activities in gills of exposed animals. A correlation between OTC- or Cu- induced changes of PKA activity and expression of hsp70 and ABCB was observed. Temperature increases (up to 24 °C) altered ABCB and hsp70 responses to the pollutants and disrupted their relationship with cAMP/PKA modulation, leading to loss of correlation between the biological endpoints. On the whole, the results indicate that temperature may impair the effects of inorganic and organic chemicals on the cAMP/PKA signaling pathway of mussels, in turn altering key molecular mediators of physiological plasticity and cytoprotection.

Tissue-scale microbiota of the Mediterranean mussel (Mytilus galloprovincialis) and its relationship with the environment.

In this study, we characterize the structural variation of the microbiota of Mytilus galloprovincialis at the tissue scale, also exploring the connection with the microbial ecosystem of the surrounding water. Mussels were sampled within a farm located in the North-Western Adriatic Sea and microbiota composition was analyzed in gills, hemolymph, digestive glands, stomach and foot by Next Generation Sequencing marker gene approach. Mussels showed a distinctive microbiota structure, with specific declinations at the tissue level. Indeed, each tissue is characterized by a distinct pattern of dominant families, reflecting a peculiar adaptation to the respective tissue niche. For instance, the microbiota of the digestive gland is characterized by Ruminococcaceae and Lachnospiraceae, being shaped to ferment complex polysaccharides of dietary origin into short-chain fatty acids, well matching the general asset of the animal gut microbiota. Conversely, the gill and hemolymph ecosystems are dominated by marine microorganisms with aerobic oxidative metabolism, consistent with the role played by these tissues as an interface with the external environment. Our findings highlight the putative importance of mussel microbiota for different aspects of host physiology, with ultimate repercussions on mussel health and productivity.

Phenotypical and molecular changes induced by carbamazepine and propranolol on larval stages of Mytilus galloprovincialis.

The possible impact of carbamazepine (CBZ) and propranolol (PROP), two widespread pharmaceuticals in the aquatic environment, were investigated on morphology and gene transcription of early larvae of Mytilus galloprovincialis. Pharmaceuticals were first tested in a wide concentration range (from 0.01 to 1000 µg/L) through the 48-hpf embryotoxicity assay. The results showed that both compounds significantly affected embryo development from environmental concentrations. Although similar EC50 were obtained, (≅ 1 µg/L) CBZ induced a progressive increase in embryo malformations, whereas PROP apparently showed greater impacts in terms of arrested development and embryo mortality at higher concentrations (>10 µg/L). Transcriptional analyses of 17 genes involved in different physiological functions in mussels and/or in their response to environmental contaminants, were performed at 24 and 48 h pf at two selected concentrations of CBZ and PROP (0.01 and 1 µg/L). Both compounds induced down-regulation of shell-specific and neuroendocrine related transcripts, while distinct effects were observed on antioxidant, lysosomal, and immune-related transcripts, also depending on the larval stage investigated. The results demonstrate that CBZ and PROP can affect development and gene transcription in mussel early larvae at environmental concentrations.

Microplastic exposure and effects in aquatic organisms: A physiological perspective.

The impact of microplastics (MPs) on aquatic life, given their ubiquitous presence in the water compartment, represents a growing concern. Consistently, scientific knowledge is advancing rapidly, although evidence on actual adverse effects is still highly fragmented. This paper summarizes the recent literature on MP impacts on aquatic organisms in an attempt to link routes of uptake, possible alterations of physiological processes, and outcomes at different levels of biological organization. Animal feeding strategies and MP biodistribution is discussed, alongside with relevant effects at molecular, cellular, and systemic level. Pathways from animal exposure to apical physiological responses are examined to define the relevance of MPs for animal health, and to point out open questions and research gaps. Emphasis is given to emerging threats posed by leaching of plastic additives, many of which have endocrine disruption potential. The potential role of MPs as substrates for microorganism growth and vehicle for pathogen spreading is also addressed.

The Multixenobiotic resistance system as a possible protective response triggered by microplastic ingestion in Mediterranean mussels (Mytilus galloprovincialis): Larvae and adult stages.

The emerging paradigm on plastic pollution in marine environments is that microsize particles (MPs) have far more subtle effects than bigger fragments, given their size range overlapping with that of particles ingested by filter-feeders. The impacts include gut blockage, altered feeding and energy allocation, with knock-on effects on widespread physiological processes. This study investigated whether ingestion of polystyrene MPs (PS-MPs) triggers protective processes in marine mussels. The Multixenobiotic resistance (MXR) system is a cytoprotective mechanism acting as an active barrier against harmful xenobiotics and a route of metabolite detoxification. Both larvae and adults were employed in laboratory experiments with different concentrations of 3-µm PS-MPs (larvae), and 3-µm and 45-µm PS-MPs (adults) matching size range of planktonic food through the mussel lifecycle. Embryos grown in the presence of 3-µm PS-MPs showed significant reduction of MXR activity and down-regulation of ABCB and ABCC transcripts encoding the two main MXR-related transporters P-glycoprotein and the Multidrug resistance-related protein, respectively. In adults, effects of PS-MPs were assessed in haemocytes and gills, which showed different modulation of MXR activity and ABCB/ABCC expression according to MP size (haemocyte and gills) or particle concentration (haemocyte). These data showed that modulation of MXR activity is part of a generalized response triggered by particle ingestion.

Transcriptional response of the heat shock gene hsp70 aligns with differences in stress susceptibility of shallow-water corals from the Mediterranean Sea.

Shallow-water corals of the Mediterranean Sea are facing a dramatic increase in water temperature due to climate change, predicted to increase the frequency of bleaching and mass mortality events. However, supposedly not all corals are affected equally, as they show differences in stress susceptibility, as suggested by physiological outputs of corals along temperature gradients and under controlled conditions in terms of reproduction, demography, growth, calcification, and photosynthetic efficiency. In this study, gene expression and induction of a 70-kDa heat shock protein (HSP70) was analyzed in five common shallow-water hard corals in the Mediterranean Sea, namely Astroides calycularis, Balanophyllia europaea, Caryophyllia inornata, Cladocora caespitosa, and Leptopsammia pruvoti. The main aim was to assess the contribution of this evolutionary conserved cytoprotective mechanism to the physiological plasticity of these species that possess different growth modes (solitary vs colonial) and trophic strategies (zooxanthellate vs azooxanthellate). Using quantitative real-time PCR, in situ hsp70 baseline levels and expression profiles after a heat-shock exposure were assessed. Levels of hsp70 and heat stress induction were higher in zooxanthellate than in azooxanthellate species, and different heat stress transcriptional profiles were observed between colonial and solitary zooxanthellate corals. On the whole, the hsp70 transcriptional response to heat stress aligns with stress susceptibility of the species and suggests a contribution of trophic strategy and morphology in shaping coral resilience to stress. Understanding these molecular processes may contribute to assess the potential effects and relative resilience of Mediterranean corals under climate change.

Uptake and transcriptional effects of polystyrene microplastics in larval stages of the Mediterranean mussel Mytilus galloprovincialis.

The widespread occurrence of microplastics (MP) in the marine environment is cause of increasing concerns about the safety of the exposed ecosystems. Although the effects associated to the MP uptake have been studied in most marine taxa, the knowledge about their sub-lethal impacts on early life stages of marine species is still limited. Here, we investigated the uptake/retention of 3-µm polystyrene MP by early stages of the Mediterranean mussel Mytilus galloprovincialis, and the related effects on gut clearance, feeding efficiency, morphological and transcriptional parameters involved in embryo-larval development. Uptake measurements were performed on larvae at 48 h, 3, 6 and 9 days post fertilization (pf) after exposure to a range of 50-10,000 particles mL-1. At all tested pf periods, treatments resulted in a significant and linear increase of MP uptake with increasing concentrations, though levels measured at 48 h pf were significantly lower compared to 3-9 d pf. Ingested MP were retained up to 192 h in larvae's gut, suggesting a physical impact on digestive functions. No change was noted between the consumption of microalgae Nannochloropsis oculata by larvae when administered alone or in the presence of an identical concentration (2000 items mL-1) of MP. The exposure to 50-10,000 MP mL-1 did not alter the morphological development of mussel embryos; however, transcriptional alterations were observed at 50 and 500 MP mL-1, including the up-regulation of genes involved in shell biogenesis (extrapallial protein; carbonic anhydrase; chitin synthase) and immunomodulation (myticin C; mytilin B), and the inhibition of those coding for lysosomal enzymes (hexosaminidase; ß-glucorinidase; catepsin-L). In conclusion, though not highlighting morphological or feeding abnormalities, data from this study revealed the onset of physical and transcriptional impairments induced by MP in mussel larvae, indicating sub-lethal impacts which could increase their vulnerability toward further environmental stressors.